This proposal represents a continuation of a long-standing research program, the general focus of which is the determination of the signal transduction pathways involved in alteration of the [Ca2+]i sensitivity of the contractile apparatus. The Specific Aims are: (1) to determine the degree to which the Ca-independent "intrinsic" tone previously documented for ferret aorta also exists in resistance vessels. The subcellular mechanisms will be investigated by determining whether myosin light chain phosphorylation or protein kinase C activity are required. The hypothesis that intrinsic tone is universally [Ca2+]i- independent will also be tested; (2) to determine the degree to which the increased [Ca2+]i sensitivity previously documented to be caused by alpha-1 agonists in ferret aorta cells involves a decreased [Ca2+]i requirement for myosin light chain phosphorylation or, conversely, the degree to which myosin light chain phosphorylation-independent mechanisms are involved; (3) to utilize a new method of monitoring free calmodulin concentrations ([CaM]f) to quantitate [CaM]f levels and to determine if [CaM]f changes during activation of intact cells. The experiments outlined involve the use of multicellular strips and freshly isolated single cells from ferret aorta as well as pressurized cannulated microvessels from the ferret coronary bed. The techniques to be used involve fluorescent probes, "digital confocal" microscopy, quantitative image analysis, beta-escin and alpha-toxin permeabilized strips, Ca2+ indicator measurements, peptide loading methods, two-dimensional polyacrylamide gels, and western blots.